This project consists of the creation of important tools for measuring T cell receptor diversity and TCR-mediated signaling, and use of these tools in both patients and experimental models of TCR diversity. The first tool is the use of high-throughput sequencing to measure the diversity of T-cell receptor rearrangements. The strategies and protocol development are ongoing and being done in collaboration with Danny Douek's lab in the NIH VRC. Finding 1: The next generation-based TCR sequencing method and techniques for analysis are completed. We applied this method to the study of patients with rare diseases of leaky severe combined immune deficiency. These patients have mutations in genes known to cause SCID, but either have a profound lymphoproliferative disorder associated with severe dermatitis, elevated IgE and eosinophilia, called Omenn syndrome, or a milder phenotype, associated with milder effects on protein activity, characterized by autoimmunity and granulomatous disease, called hypomorphic SCID. We found that patients with Omenns syndrome due to mutations in recombinase activating gene (Rag) had marked repertoire constriction, substantially smaller CDR3 regions and impaired N-nucleotide addition compared to those with Omenn syndrome due to other causes, suggesting that normal Rag function is critical for T-cell receptor junctional diversity. We also found that while the TCR repertoire diversity in the hypomorphic SCID patients was similar to controls, and while amino acid usage was similar to controls in Omenn Syndrome patients, there were significantly different residues used in the hypomorphic SCID, arguing for a specific set of TCRs which are pathogenic in their disease and not in a disease characterized by even more profound repertoire reduction. The results of this study were published in the Journal of Allergy and Clinical Immunology. Our findings were validated in a companion manuscript in the Journal of Allergy and Clinical Immunology from the Notarangelo lab showing similar results in an in vitro B-cell receptor recombination system, interrogating the various mutations in Rag. We further attempted to interrogate the sequences of patients with graft-vs-host disease soon after transplant to see if they correlated to any sequences found in our patient cohort. No overlap was found.